PRTC preparation

PRTC: We know what those peptides can be

This is the last step in the lab before we go on the mass spectrometer! PRTC, or Peptide Retention Time Calibration, is a mix of known peptides and known quantities that we can use to ensure the mass spectrometer is functioning. It also provides a reference for our sample analysis.

Diluting PRTC:

The entire preparation step requires pipetting small volumes of liquids. To reduce the capacity for human error, we diluted the 0.5 pmol/µL stock solution of PRTC so we would not have to pipet any volume less than 1 µL.

Preparing samples:

Our goal is to extract 3 µL of our prepared sample for each mass spectrometer replicate, which means we need 6 µL of sample total. To prepare for this, our total volume of sample and solvents will be 15 µL.

Removed samples from -80ºC

Placed in wet ice bath and allow to thaw

Labelled glass autosampler vials, one for each sample. These vials are the ones we will put in the mass spectrometer

Labelled new centrifuge tubes, one for each sample

To each newly labelled sample centrifuge tube, we added the following reagents

3.13 µL of Final Solvent

10 µL of sample removed from -80ºC

1.88 µL of 0.2 pmol/µL PRTC

Because we pipetted such small quantities of PRTC, we ensured the pipet tip was touching the bottom of the centrifuge tube so all liquid was transferred

Vortexed gently to thoroughly mix contents

Centrifuged liquid down to the bottom of the centrfuge tube

After we prepared the samples with PRTC, we gave everything to Emma, who put the samples in her lab’s -80ºC. When we are ready to use the mass spectrometer, we will transfer the contents of the centrifuge tubes to the labelled glass autosampler vials.